The antigen-antibody reaction is the basis for all immunological test methods. Special proteins called antibodies are produced by an animal in response to the presence of an antigen, that is a foreign protein, in the body fluids of the animal. This normal body response to a foreign protein has lead to the development of a number of techniques which are used to diagnose various human and animal diseases or disorders. Immunological test methods may also be used to detect pregnancy. In vitro tests for the presence of a suspected antigen or antibody in a body fluid are carried out by adding the immunological counterpart to a vial of the body fluid, i.e. add antigen if the test is for the presence of antibody or add antibody if the test is for the presence of antigen. If the suspected protein is present the resulting antigen-antibody reaction is generally manifested by precipitation or agglutination of the antigen-antibody complex. As used herein the term body fluid refers to urine, serum, plasma, or the like.
In some instances the antigen-antibody complex is slow to form and the particles that are formed are too small to be observed with certainty. In such cases, detectability of the antigen-antibody reaction can be improved by utilizing a carrier. When the antigen or antibody is coated on the surface of a carrier the reaction with the immunological counterpart produces a visible mass or agglutant. The proteinic antigen or antibody may be adsorbed onto the surface of carriers such as erythrocytes, bacterial cells, bentonite, polystyrene latex particles, anionic phenolic resins, or finely divided diazotized amino cellulose. It has been found however, that chemical binding of the antigen or antibody molecule to the carrier is superior to physical adsorption. U.S. Pat. No. 3,857,931 teaches that proteinic antigens or antibodies can be chemically bound to a polymer latex carrier having surface carbonyl groups. Amide bonds form between the protein and carboxylated latex in the presence of a water-soluble carbodiimide coupling agent.
Mechanistically, the carbodiimide is believed to react with the carboxyl groups on the latex to form a transient activated intermediate acylisourea which in turn reacts with the amino groups on the protein to form a stable amide board affixing the protein to the latex surface. See DeTar, D. F., et al., J. Amer. Chem. Soc. 88, 1024 (1966).
A disadvantage of processes used by the prior art is the inability to control the undesirable and indiscriminate reaction of the carbodiimide with the carbonyl groups that are also present on the proteinic antigens and antibodies. Thus, the activated carbonyls on the protein itself can react with the amino group of the protein resulting in intra- or inter-protein crosslinking. This can result in conformational or structural distortion of the protein molecule which in turn can effect immunochemical sensitivity. These side reactions are especially acute when the protein contains relatively large amounts of aspartic and glutamic acid in the protein chain since these amino acids contain free carboxyl groups. An example of such a protein is human chorionic gonadotropin, hereafter called HCG, which has an isoelectric point of 2.95, indicative of a high negative charge.
A number of immunological tests have been developed for the detection of HCG in urine or serum. HCG is a glycoprotein with a molecular weight of about 27,000 produced by the chorionic tissue of the placenta during pregnancy. Systems using HCG have been used to detect pregnancy. During pregnancy this hormone governs the production and secretion of progesterone by the corpus luteum. HCG is also produced in large quantities by hydatidiform moles, choriocarcinomas, and some tumors of the testis. Low levels of HCG have also been found by radioimmunoassay in the sera of patients with various nontrophoblastic neoplasms. Various agglutination techniques have been used to test for the presence of HCG.
Agglutination testing for HCG may be performed by either the indirect or the direct technique. In the indirect technique the clinical sample is mixed with HCG antibody at a dilution that will be completely bound by one or more I.U./ML HCG. After an initial incubation period an indicator system consisting of HCG bound to a particulate carrier (latex or red cells) is added to the mixture. If HCG is present in the clinical sample the HCG antibody will not be available to react with the HCG-carrier complex and there will be no agglutination, thus, absence of agglutination is a positive test for HCG. If, on the other hand, HCG is not present in the clinical sample the HCG antibody will react with the HCG-carrier complex causing agglutination of the indicator system. This is a negative test for HCG in the clinical sample. In the direct technique HCG antibody bound to the carrier reacts directly with the HCG in the clinical sample and there is no need for an intermediate incubation step. Thus, in the direct technique agglutination indicates a positive test for HCG in the clinical sample.
Chemical coupling of protein to latex particles by means of an amide bond is discussed in Am. Review of Biochemistry, 35 (II), 896 (1966) and in Ohno, Y. Water-Insoluble Acrylic Polymer Derivatives of Enzymes and Antigens, The Univ. of Wisc. (1967). Chemical coupling of a protein to a carrier for use in immunological diagnosis is shown in U.S. Pat. Nos. 3,533,310; 3,775,536; and 3,879,262.